One of important abilities of proteins is to specifically bind to a particular molecule. Due to this ability, some proteins play important roles in immunoreactions or signal transduction in vivo. Methods for separation and purification of useful substances utilizing this ability are also being actively developed. One example of such methods actually used in industrial applications is Protein A affinity separation matrices that are used to purify (capture) antibody drugs from animal cell cultures at one time at high purity levels.
Antibody drugs developed so far are generally monoclonal antibodies, which are mass-produced by recombinant cell culture techniques or the like. The term “monoclonal antibodies” refers to antibodies that are produced by clones of a unique antibody-producing cell. Almost all the antibody drugs currently available on the market are classified into immunoglobulin. G (IgG) subclasses based on their molecular structures. Protein A is a cell wall protein produced by the gram-positive bacterium Staphylococcus aureus, and contains a signal sequence S, five immunoglobulin-binding domains (B domain, D domain, A domain, B domain, and C domain), and a cell wall-anchoring domain known as XM region (Non Patent Literature 1). In the initial purification step (capture step) in the manufacture of antibody drugs, affinity chromatography columns obtained by immobilizing Protein. A as a ligand on a water-insoluble carrier are commonly used (Non Patent Literatures 1 to 3).
Various techniques for improving the performance of Protein A columns have been developed. Technological developments in ligands are also being made. Initially, wild-type Protein A has been used as a ligand, and currently, Protein A variants that have been modified by protein engineering are used as ligands in many techniques for improving the column performance. Notably, some of the Protein. A engineering techniques developed so far focus on now to immobilize Protein A ligands on water-insoluble carriers.
Proteinic ligands including Protein A are each immobilized on a carrier at multiple sites by covalent bonding of the reactive side chain functional groups of lysine (Lys) or cysteine (Cys) residues (which are not present in Protein. A) in the proteins in a manner shown in FIG. 1(1) (Non Patent Literature 4). A recombinant Protein A which has been modified to have a different ratio between the number of lysine (Lys) residues on the antibody binding face and the number of lysine (Lys) residues on the non-binding face of Protein A (Patent Literature 1) is a technique allowing effective use of the antibody binding domains of the ligand even though it has basically the same immobilization form as shown in FIG. 1(1).
Protein A variants that are Protein A containing a mutation of one cysteine (Cys) residue (Patent Literature 2 and Non Patent Literature 5) are each site-specifically immobilized on a carrier via the Cys residue. Protein A variants containing complete deletions of Lys or Cys residues in the amino acid sequence (Patent Literatures 3 and 4) are techniques that immobilize the protein on a carrier via the N terminal (α-amino group) or C terminal (special tag). These techniques allow a protein ligand to be immobilized on a carrier via the terminal in a manner shown in FIG. 1(2) or (3), and the advantages of these techniques are that the orientation of the ligand can be controlled and that the carrier surface area can be effectively used, as mentioned in these documents.
Thus, developments in techniques for immobilizing a protein ligand to an affinity separation matrix are mainly based on techniques relating to Protein A columns that are required to have high performance because of their high industrial usefulness.